The type 2 iodothyronine deiodinase (D2) is a pivotal regulatory selenoenzyme catalyzing the first step in thyroid hormone action, the conversion of thyroxine (T4) to the active hormone, 3,5,3'-triiodothyronine (T3). Studies supported by this proposal have led to the novel conclusion that the activity of this enzyme is regulated post-translationally by a dynamic balance between ubiquitination, causing D2 inactivation and proteasomal degradation, and deubiquitination, which reactivates it. Our goals are to identify the proteins and regulatory steps in these two processes. D2 is an endoplasmic reticulum (ER) integral membrane protein selectively ubiquitinated and targeted to the proteasomal system, a process termed ER-associated degradation (ERAD). We identified the ERAD-related UBC6 and UBC7 as important ubiquitin conjugases (E2s) for D2. We hypothesize that D2 ubiquitination is initiated after recognition by an as yet unidentified D2-specific ubiquitin ligase or ligases (E3s) that associate with these E2s. Specific Aim I will evaluate E3 candidates isolated from human brain, their D2 and E2 contact sites and their function. D2 is constantly ubiquitinated under basal conditions but this is accelerated by T4 catalysis. The critical steps in these processes are unknown. Is substrate-induced ubiquitination a similar, but faster, reaction than basal ubiquitination or are there different sets of E2s or D2-specific E3s involved? Since the events we are investigating are complex, Specific Aim II will use in vivo approaches including mammalian two-hybrid systems and peptides, which can block E3-D2 binding to confirm the in vitro results and define T4-related events. Using a yeast two-hybrid system, we identified two closely related D2-binding ubiquitin-specific processing proteases (UBPs) in human brain that deubiquitinate D2 thus reactivating it. In brown adipose tissue, cold exposure of rodents (a paradigm for events during human parturition) induces a rapid increase in one of these. This deubiquitinates D2 providing active enzyme more rapidly than does de novo synthesis. Little is known of these only recently identified UBPs and Specific Aim III will define their D2 interaction mechanism, developmental expression and regulation by T3, cAMP and other hormones. With these studies, we hope to place this newly recognized mechanism for maintaining T3 homeostasis into a physiological context.